1. Field of the Invention
The present invention relate to a peptide for binding thereto a low density lipoprotein (hereinafter, frequently referred to simply as an "LDL") and an adsorbent comprising a water-insoluble carrier having bonded thereto the peptide. More particularly, the present invention is concerned with a peptide for binding thereto an LDL, wherein the peptide has a specific amino acid sequence comprising 2 to 10 amino acid residues including at least one Phe or Trp and at least one Arg or Lys and wherein the peptide has a specific electric charge (E) which is defined by the formula: E=(the number of positive functional groups present in the peptide)-(the number of negative functional groups present in the peptide). Further, the present invention is also concerned with an adsorbent for removing an LDL from a body fluid, comprising a water-insoluble carrier having bonded thereto the peptide. Still further, the present invention is also concerned with a method for removing an LDL from a body fluid, comprising contacting the body fluid with the peptide.
The peptide of the present invention is advantageous in that the peptide not only has an excellent ability to specifically bind thereto an LDL, but also is free from difficult problems, such as the production of a bradykinin, the activation of blood cells, the adsorption of blood cells onto the peptide and the activation of a blood coagulation system, thus leading to a safety in use of the peptide. Therefore, the peptide of the present invention can be advantageously used not only as a reagent for adsorption-removing an LDL from a body fluid, such as whole blood and plasma, but also as a peptide drug or a carrier peptide for a drug for treating a disease caused by an LDL. Further, the peptide of the present invention, which has the ability to bind thereto an LDL, has only 10 amino acid residues or less and, hence, is advantageous not only in that it can be easily prepared at low cost, but also in that it has excellent stability, such as sterilization stability and storage stability. Further, when an adsorbent comprising a water-insoluble carrier having bonded thereto the peptide of the present invention is employed in a blood purification treatment device or the like (which is necessarily used for removing the LDL from the blood of a patient suffering from a disease in which, due to a morbid factor, the LDL concentration of the blood is caused to increase to a level higher than that of the LDL concentration of the blood of a healthy person), the LDL can be efficiently, safely removed to advantage on the patient. In addition, when a soft gel (such as an agarose gel) or a hard gel (such as cross-linked polyvinyl alcohol) is used as the above-mentioned water-insoluble carrier in the adsorbent, the adsorbent can be advantageously used as a gel in liquid chromatography and the like for separating from an LDL-containing liquid the LDL in high purity form.
In the present specification, amino acid residues are represented using abbreviations, as indicated below, approved by IUPAC-IUB Commission on Biochemical Nomenclature (CBN). With respect to amino acids and the like having isomers, those which are represented by the following abbreviations are of either L-form or D-form. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.
A or Ala: alanine residue PA0 D or Asp: aspartic acid residue PA0 E or Glu: glutamic acid residue PA0 F or Phe: phenylalanine residue PA0 G or Gly: glycine residue PA0 H or His: histidine residue PA0 I or Ile: isoleucine residue PA0 K or Lys: lysine residue PA0 L or Leu: leucine residue PA0 M or Met: methionine residue PA0 N or Asn: asparagine residue PA0 P or Pro: proline residue PA0 Q or Gln: glutamine residue PA0 R or Arg: arginine residue PA0 S or Ser: serine residue PA0 T or Thr: threonine residue PA0 V or Val: valine residue PA0 W or Trp: tryptophan residue PA0 Y or Tyr: tyrosine residue PA0 C or Cys: cysteine residue
2. Prior art
It has been known that, among the lipoproteins present in blood, an LDL contains a large amount of cholesterol and therefore is causative of arteriosclerosis. Conventionally, the treatment of a patient suffering from familial hypercholesterolemia, in which the LDL concentration of the blood of the patient is high, is conducted by extracorporeal blood circulation therapy (hereinafter, frequently referred to as an "LDL apheresis") using an adsorbent having the ability to adsorb an LDL, with the result that various symptoms of the patient have been ameliorated.
With respect to the known adsorbents for removing the LDL from blood, for example, European Patent No. 0 225 867 (corresponding to Examined Japanese Patent Application Publication Nos. 62-56782 and 63-19214) discloses a resin on which a polysaccharide sulfate having a negative charge is chemically immobilized as a ligand. As an example of such an adsorbent, there can be mentioned a cellulose particle carrier having dextran sulfate immobilized thereon as a ligand, which is commercially available.
However, when the LDL apheresis is conducted using such an LDL adsorbent having dextran sulfate as a ligand, a physiologically active substance called bradykinin is disadvantageously produced in the blood. The bradykinin is known to cause blood pressure depression, the contraction of a smooth muscle, the membrane-permeability sthenia and the like (see, for example, European Patent Application Publication No. 0 561 379 A1 (European Patent Application No. 93 104 348.3).
Further, WO90/04416 discloses an agarose particle on which the antibody having the ability to bind thereto a human low density lipoprotein is immobilized.
However, the use of an antibody in an LDL adsorbent has problems as follows. The antibody is generally produced in vitro or in vivo. Therefore, for preparing the antibody in an amount sufficient to treat a patient suffering from a disease caused by an LDL, large amounts of labor and cost are disadvantageously needed. Further, the antibody disadvantageously exhibits poor stability, especially in the sterilization thereof, so that the antibody cannot be safely used in an extracorporeal blood circulation therapy.
As further examples of substances suggested to have the ability to bind thereto an LDL, there can be mentioned a polylysine and a polyarginine, each having about 25 to 250 amino acid residues {see Olov Wiklund et al.; Cationic polypeptides modulate in vitro association of low density lipoprotein with arterial proteoglycans, fibloblasts, and arterial tissue. Arteriosclerosis. 10, 695-702 (1990)}; and a peptide having 15 amino acid residues of VVWRLTRKRGLKVVV (see Urban Olsson et al.; Binding of a synthetic apolipoprotein B-100 peptide and peptide analogues to chondroitin 6-sulfate: Effects of the lipid environment. Biochemistry, 1993, 32, 1858-1865).
However, each of the above-mentioned polypeptides (i.e., the polylysine and the polyarginine, each having about 25 to 250 amino acid residues, and the polypeptide containing 15 amino acid resides of VVWRLTRKRGLKVVV) has more than 10 amino acid residues. Therefore, as in the case of the antibody, these polypeptides are poor with respect to stability, such as the sterilization stability and the storage stability.
On the other hand, with respect to a substance having a very high positive charge, it is known that when blood contacts with the surface of such a substance, disadvantages are caused such that the cell components of blood (such as erythrocytes, leukocytes and platelets) are activated and that the cell components are adsorbed on the surface of the above-mentioned substance due to a strong electrostatic interaction therebetween. Further, such a substance disadvantageously induces unfavorable phenomena, such as a non-specific adsorption of plasma proteins on the substance and the activation of a blood coagulation factor. Therefore, from the viewpoint of safety, it is not preferred to use the above-mentioned substance as an adsorbent in the LDL apheresis therapy.
Therefore, it has been desired to develop a new substance for binding thereto an LDL, which not only has an excellent ability to specifically bind thereto an LDL, but also does not cause disadvantages, such as the production of bradykinin, the activation of blood cells, the adsorption of blood cells on the substance and the activation of a blood coagulation system and which, therefore, can be advantageously used in an adsorbent for removing an LDL from a body fluid, such as whole blood or plasma.